Pulmonary-Renal Syndrome from Levamisole-Adulterated Cocaine-Induced Antineutrophil Cytoplasmic Antibody (ANCA)-Associated Vasculitis: A Systematic Review

Levamisole is an anti-helminthic drug with immunomodulatory properties that is added to cocaine to increase its potency and weight. Levamisole-adulterated cocaine (LAC) may cause an antineutrophil cytoplasmic antibody (ANCA)-associated systemic small vessel vasculitis (AAV). We aimed to characterize the phenotype of persons developing pulmonary-renal syndrome (PRS) in LAC-induced AAV and summarize its treatment and outcomes. Pubmed and Web of Science were searched (until September 2022). Reports that described co-existing diffuse alveolar hemorrhage and glomerulonephritis in an adult (age ≥ 18) with confirmed or suspected LAC exposure were included. Reports, demographics, clinical and serologic features, treatment and outcome characteristics were extracted. Of the 280 records identified, eight met the inclusion criteria, including eight unique cases. Persons were aged 22–58 years, and 50% were women. Cutaneous involvement occurred in only half of the cases. Other associated vasculitis findings and serologies were heterogeneous. All patients received immunosuppression with steroids, with cyclophosphamide and rituximab commonly added. We concluded that PRS could occur from LAC-induced AAV. Distinguishing LAC-induced AAV from primary AAV is challenging as clinical and serologic presentations overlap. Asking about cocaine use is requisite in persons presenting with PRS to guide diagnosis and appropriately counsel on cocaine cessation in conjunction with immunosuppression as treatment.


Introduction
Levamisole is an anti-helminthic drug with immunomodulatory properties that was previously used in the treatment of various medical conditions, including autoimmune disorders and as an adjuvant chemotherapy agent for cancer [1]. The therapeutic use of levamisole in humans has been discontinued in many countries due to severe adverse effects, including agranulocytosis, seizures, skin necrosis, and vasculitis [2][3][4]. A systemic, antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis has been reported in patients on therapeutic doses of levamisole [5][6][7]. Levamisole remains widely available as a veterinary medicine and is commonly used as a cutting agent for cocaine to increase its potency and weight [3,8]. In 2019, the crude global prevalence of cocaine use disorder was 0.06% (95% confidence interval, 0.04-0.07%) [9]. Therefore, an estimated 308 to 539 million persons globally are regularly using cocaine [9,10]. The prevalence of cocaine use disorder is highest in the Americas and Europe [9]. In these high-use regions, levamisole has been detected as an adulterant in 52-69% of cocaine samples systematically surveyed [11,12], including glucocorticoids, a choice of rituximab or cyclophosphamide, and consideration of the addition of the C5a receptor inhibitor, avacopan [4,15,33,34].
The purpose of this literature review was two-fold: [1] to characterize the phenotype of persons who develop PRS secondary to LAC-induced AAV, and [2] to summarize the treatments employed and their outcomes in known cases of PRS secondary to LACinduced AAV.

Methods
Two primary databases (PubMed and Web of Science) were searched from inception until 2 September 2022 using the following keywords: ("levamisole" OR "cocaine") AND "pulmonary-renal syndrome" OR "glomerulonephritis" OR "alveolar hemorrhage" OR ("vasculitis" AND "kidney") OR ("vasculitis" AND "lung") OR ("vasculitis" AND "pulmonary") OR ("vasculitis" AND "renal"). The search was limited to studies written in English and involving human subjects. Additional citations were added through a Google Scholar search and review of references from included reports. Case reports and case series were eligible for inclusion if they [1] reported at least 1 case of pulmonary-renal syndrome (PRS; defined as co-existing DAH and glomerulonephritis) and the case of interest was reported in [2] an adult (aged at least 18 years) with [3] confirmed or suspected levamisoleadulterated cocaine (LAC) exposure, and [4] a positive ANCA serologic test. Studies were excluded if they reported a case [1] in a pediatric patient (aged < 18 years), [2] the case only described pulmonary or renal vasculitis manifestations, but not both occurring simultaneously, or [3] there was insufficient evidence reported to confidently conclude the presence of DAH or glomerulonephritis as the etiology of the pulmonary or renal involvement, respectively.
One author (P.B., M.W., or R.A.) individually screened each article (title, keywords and abstract), and subsequently, the full texts of records recognized as potentially eligible by abstract screening. A second reviewer and subject matter expert (R.E.E.) was included to resolve discrepancies. Predetermined fields for data extraction from each report included demographics (age, sex, race, ethnicity), the time between onset/diagnosis of vasculitis and onset of PRS manifestation, the method of ascertainment of levamisole exposure, evidence of DAH, evidence of glomerulonephritis, cutaneous manifestations of vasculitis (if present), other organ system manifestations of vasculitis (if present), ANCA profile and other serologies, renal indices, treatment(s) described, and outcome(s) of the case.
This study was reviewed by the Augusta University Institutional Review Board and determined not to be research involving human subjects.

Results
Two hundred eighty records were identified from the two primary databases. Thirtyfive additional records were identified through a Google Scholar search and review of references from included reports. Of these records, eight met the inclusion criteria. A flow chart of the literature search strategy is depicted in Figure 1. Each included article identified detailed one unique case of PRS secondary to LAC-induced AAV. Six articles were full-text manuscripts, and two were published abstracts. The characteristics of the eight identified cases of PRS secondary to LAC-induced AAV are summarized in Table 1. Serologic profiles and renal indices at the time of PRS presentation in the included cases are detailed in Table 2.

Patient Characteristics
Age ranged from 22 to 58 years with a mean age of 41 years (standard deviation 11.7 years). Half of the cases occurred in men and half in women. Race and ethnicity were reported very infrequently. The majority of cases (63%) presented with PRS as the initial manifestation of LAC-induced AAV, but others reported PRS onset after a long-standing LAC-induced AAV diagnosis. Two of the eight cases reported a pre-existing autoimmune diagnosis in the past medical history: one patient had a history of reactive arthritis [31], and another had a history of systemic lupus erythematosus [32]. Half of the cases determined levamisole exposure based on cocaine use by history, and the other half of cases confirmed cocaine positivity on urine drug screening. Only one case further confirmed levamisole in a urine sample by liquid chromatography-mass spectrometry [27].

Clinical and Pathologic Features
Most cases (75%) confirmed DAH with bronchoscopy, but two cases diagnosed DAH based on hemoptysis and compatible chest imaging without bronchoscopy. Renal biopsy confirmed glomerulonephritis in seven cases (88%). The remaining case was suggestive of glomerulonephritis by reporting the presence of hematuria and proteinuria on urinalysis [29]. Of cases with renal biopsy, rapidly progressive renal disease was supported by the presence of either necrosis or crescents in six of seven cases (86%). Immunofluorescence of renal biopsies was variable, with two cases reporting pauci-immune RPGN and two cases reporting immune-complex mediated disease. Two cases reported concomitant membranous nephropathy.
The most commonly reported associated organ involvement was otolaryngologic (63%), with epistaxis and destruction/perforation of nasal septum reported. Cutaneous and musculoskeletal involvement each occurred in 50% of cases. Two cases (25%) had both arthralgia and skin lesions consisting of erythema to purpura/necrosis on the face and extremities [25,29]. The most common skin biopsy finding reported was leukocytoclastic vasculitis. Constitutional symptoms were found in three cases (38%), and each of the following vasculitis features was reported once: testicular vasculitis, venous thrombosis, and heart failure. One case had no reported associated vasculitis features outside of pulmonary and renal involvement as PRS.

Serologic and Other Laboratory Findings
The majority of cases were positive for myeloperoxidase [17] antibody (63%). When the ANCA pattern was reported, these patients with positive MPO antibodies demonstrated typical perinuclear ANCA (P-ANCA) positivity as well (three of five cases). Two cases were proteinase-3 (PR3) antibody positive (25%). One case was PR3 positive in the setting of high titer cytoplasmic ANCA (C-ANCA) positivity, negative P-ANCA and negative MPO antibody [30]. The other was dual PR3 and MPO antibody positive [31]. One case was low-titer positive for P-ANCA (1:80) and negative for C-ANCA, PR3, and MPO. Positive APLA, ANA, dsDNA, rheumatoid factor (RF), Anti-Ro, and Anti-La were all reported, but none consistently across more than two studies. In one published abstract, ANCA serologies were not directly reported, but this patient had a history of LAC-induced AAV, and the authors state, "workup suggested a recurrence of [LAC-induced AAV] in the setting of recent cocaine use" [32].
Creatinine at presentation with PRS varied widely from normal to very high (up to 11.07 mg/dL). When urinalysis results were reported in six of eight cases, both proteinuria and hematuria were present. Half of these cases reported nephrotic range proteinuria, one case reported non-nephrotic range proteinuria, and the remaining two cases did not specify the amount of proteinuria. Dysmorphic and crenated red blood cells were reported on urine microscopy.

Treatment and Outcomes
All eight of the cases reported were treated with high-dose steroids. Six of eight cases received additional immunosuppression with cyclophosphamide (63%) and/or rituximab (25%). One person who initially received IV cyclophosphamide was transitioned to maintenance azathioprine therapy but did not tolerate this medication [27]. The maintenance of the immunosuppressive therapy regimen was not reported in any other case. Three persons required hemodialysis (38%), two patients received plasmapheresis (25%), and one person received IV recombinant activated factor VII (rFVIIa) for refractory DAH. Two reported cases of PRS in LAC-induced AAV remained dialysis-dependent, but the remaining cases reported at least partial recovery. The third case reporting hemodialysis as an acute intervention did not report the renal outcome.

Discussion
Pulmonary-renal syndrome (PRS) can occur from levamisole-adulterated cocaineinduced antineutrophil cytoplasmic antibody-associated vasculitis (LAC-induced AAV). In this literature review, we determined that PRS from LAC-induced AAV is gender indiscriminate and highly heterogeneous in phenotype with regards to age, associated clinical features and analytical results, which may lead to diagnostic and treatment difficulties.
The diagnosis of LAC-induced AAV is a diagnosis of exclusion based on compatible clinical presentation in the context of a reasonable ascertainment of exposure to levamisoleadulterated cocaine. The classic presentation of LAC-induced AAV includes fever, arthralgia, otolaryngologic symptoms, and, in the vast majority of cases, cutaneous involvement, most commonly as retiform purpuric lesions with or without central necrosis, with a special predilection for the face and earlobes [14,15,35]. Typical pathologic lesions on skin biopsy are leukocytoclastic vasculitis and/or thrombotic vasculopathy with the involvement of superficial and deep dermal vessels [35,36]. In this review, these usual findings of LAC-induced AAV that may prompt the rheumatologist or other clinician to prioritize a LAC-induced AAV diagnosis as the potential cause of PRS were not consistently described. While skin involvement is seen in over 90% of cases of LAC-induced vasculitis [15,37], only 50% of cases of PRS secondary to LAC-induced vasculopathy in this review reported any associated cutaneous lesions. Further, PRS occurred as the presenting symptom of LACinduced AAV in the majority of the cases described. Clinical symptoms alone are unlikely to help differentiate PRS from LAC-induced AAV versus primary and other secondary forms of AAV.
Antineutrophil cytoplasmic antibodies (ANCA), autoantibodies directed against antigens in the cytoplasmic granules of neutrophils and monocytes, may be helpful in distinguishing types of ANCA-associated vasculitis (AAV) with often overlapping clinical presentations [38]. Granulomatosis with polyangiitis typically displays a pattern of cytoplasmic ANCA (C-ANCA) almost exclusively as a result of antibodies directed against proteinase-3 (PR3), while microscopic polyangiitis typically shows a pattern of perinuclear ANCA (P-ANCA) targeting myeloperoxidase [17,38]. The ANCA pattern that characterizes LAC-induced AAV is a positive P-ANCA with or without positive MPO titers [14,36]. A potential discordance between P-ANCA and MPO in some cases of LAC-induced AAV has been attributed to P-ANCA production directed at other neutrophil granule components such as human neutrophil elastase (HNE), lactoferrin, cathepsin G, and sometimes PR3 [36,[38][39][40]. P-ANCA targeting HNE is reported in cocaine-induced midline destructive lesions and LAC-induced cutaneous vasculopathy but is notably absent in primary AAV [40]. Combined positivity for MPO antibody and PR3 antibody is reported in druginduced AAV, including LAC-induced AAV, but is uncommon in primary AAV [14,15,41]. HNE and cathepsin G share epitopes with PR3, which may also be responsible for the combined positivity for MPO and PR3 antibodies through cross-reactivity in LAC-induced AAV [26]. In addition, patients with LAC-induced AAV are often positive for other autoantigens, including ANA, APLA, anti-dsDNA, and others [3,4,14,15].
There are limited reports of levamisole-induced AAV in the absence of concomitant cocaine exposure [5][6][7]35,42], and none, to our knowledge, report pulmonary or renal vasculitis secondary to isolated levamisole exposure. The exact pathogenesis of LAC-induced AAV is unclear, but several potential mechanisms have been described. Levamisole has been shown to cause immune system dysregulation that could provoke an autoimmune vasculitis in genetically predisposed individuals through various mechanisms: formation of antibodies with increased B and T cells, chemotaxis and increased neutrophil response, deposition of immune complexes in blood vessel walls, and degranulation and release of oxygen metabolites leading to vascular injury [35]. Importantly, both cocaine and levamisole induce the formation of neutrophil extracellular traps (NETs) enriched in neutrophil elastase, a potential target for ANCAs and a mechanism for loss of self-tolerance in individuals who develop LAC-induced AAV [43]. Both cocaine and levamisole also increase the release of B-cell activating factor (BAFF), which may promote the survival and differentiation of B cells producing pathologic ANCAs in LAC-induced AAV [43].
In this review, our findings in patients with PRS secondary to LAC-induced AAV were consistent with existing literature regarding serologic profiles of LAC-induced AAV in general. However, the serologic profiles reported were non-uniform. The most commonly seen ANCA serologic profile was P-ANCA positivity without or without MPO antibody; however, this accounted for only half of the cases. Dual PR3 and MPO antibody positivity was reported in only one case. One case displayed typical C-ANCA targeting PR3-positivity indistinguishable from the typical ANCA profile of granulomatosis with polyangiitis. Positive APLA, ANA, dsDNA, RF, Anti-Ro, and Anti-La were all reported, but none consistently across more than two studies. No serologic features predominated in this review that would consistently distinguish PRS secondary to LAC-induced AAV from PRS secondary to primary AAV. Anti-HNE was not reported in any of the cases in our review. Lood and Hughes have demonstrated that the pathogenesis of LAC-induced AAV may be driven by the ability of cocaine and levamisole to induce the release of inflammatory neutrophil extracellular traps (NETs), leading to neutrophil elastase (HNE) autoantigen production [43]. Systematic ascertainment of anti-HNE may be an important future direction that deserves clinical attention in future series of LAC-induced AAV with PRS as this could be a means of distinguishing LAC-induced AAV from primary AAV.
Levamisole was confirmed in the urine by liquid chromatography-mass spectrometry (LC-MS) in only one of eight cases of PRS due to LAC-induced AAV in this review. Instead, cocaine exposure was determined either by history alone or with a conventional urine drug screen. The pharmacokinetics of levamisole, with a short plasma elimination half-life of 5.6 h, extensive metabolism, and minimal unchanged drug urinary excretion (2-5%), make it a difficult agent to detect in biological samples [44,45]. Clinicians treating patients presenting with PRS must have a high index of suspicion for LAC-induced AAV early as the window for confirmatory testing for urinary levamisole with LC-MS is very narrow. However, given the high rates of adulteration of cocaine with levamisole [11][12][13], confirmation of levamisole in urine samples is likely unnecessary if a thorough and detailed social history is undertaken to ascertain a history of regular cocaine exposure when possible. When history is limited due to intubation or otherwise, a conventional urinary drug screen positive for cocaine would still be highly suggestive of levamisole-adulterated cocaine exposure.
More information is needed about the optimal treatment of PRS secondary to LACinduced AAV. The existing paradigm of treatment for LAC-induced AAV, in general, has been a complete cessation of cocaine (and thereby, levamisole) use [14,35,46]. In the absence of internal organ involvement, additional immunosuppression may be unnecessary, although relapse rates are high with the reintroduction of cocaine [15,37]. Corticosteroids are commonly used in life and/or organ-threatening LAC-induced AAV and were given to all patients with a manifestation of PRS in this review [15,26,37]. Cyclophosphamide or rituximab are the most commonly reported additional immunosuppressants used to treat PRS secondary to LAC-induced vasculitis, which is in accordance with existing clinical practice guidelines for the treatment of primary AAV with active severe (life-or organthreatening manifestations) disease [33] and existing reports for other severe LAC-induced vasculitis cases [15,26]. Rituxan is a much newer agent compared to cyclophosphamide to treat adult patients with granulomatosis with polyangiitis and microscopic polyangiitis in adults in combination with glucocorticoids (U.S. Food and Drug Administration approved since 2011) [47], which may or may not account for more use of cyclophosphamide in this review.
Plasmapheresis was used twice in these cases, once with reported recovery in a patient with the primary manifestation of DAH over renal involvement and once in a patient who required hemodialysis and remained dialysis-dependent at the time of publication. In both instances, the cases occurred before the PEXIVAS trial in 2020 demonstrated no reduction in death or kidney failure with the use of plasmapheresis in persons with severe primary AAV [48]. However, in our review, immune complex deposition was more regularly reported on skin and renal biopsies than in classic primary AAV, so the role of plasmapheresis in LAC-induced AAV remains unclear. For refractory DAH, IV recombinant activated factor VII was used in one case with a favorable outcome. There is a paucity of data on maintenance therapy for PRS secondary to LAC-induced AAV. Only a single case in this review reported on any maintenance therapy being employed, and the agent used was azathioprine, with no data on the outcome of its use as the patient did not tolerate the azathioprine [27]. Dialysis dependence is unfortunately not uncommon in patients with PRS from LAC-induced AAV per this review, as has also been reported in other cases of isolated RPGN from LAC-induced AAV [26,49,50].
This review has limitations. The data on evidence for DAH, evidence for glomerulonephritis, or associated vasculitis symptoms may have been incompletely reported in manuscripts or published abstracts, leading to incomplete case ascertainment or lack of detail to inform clinical phenotypes in this review. Data on ANCA profiles was incomplete in several included published reports which may have impaired our ability to characterize the serologic profile of PRS in LAC-induced AAV. We were unable to distinguish in each case with otolaryngologic involvement whether these manifestations were due to LACinduced AAV or direct toxicity of cocaine, i.e., CIMDL, given their substantial overlap in clinical presentation, as is often the case in clinical practice. With the exception of Pecci et al. [29], where bronchial biopsy confirmed pulmonary vasculitis, we were unable to definitively attribute DAH in these cases to LAC-induced AAV rather than a direct toxic effect of smoked cocaine-induced intense vasoconstriction. However, the concomitant presence of RPGN reasonably suggests systemic vasculitis as the etiology of PRS in these cases, as RPGN is not a reported feature of cocaine-induced pathology in the absence of vasculitis [22]. Levamisole exposure was not confirmed by LC-MS in urinary samples in most cases. Finally, publication bias may influence the phenotypes of PRS secondary to LAC-induced AAV published and subsequently described here.

Conclusions
Asking about cocaine use is requisite in persons presenting with PRS. PRS may be due to LAC-induced AAV. Differentiation of PRS secondary to LAC-induced AAV versus primary and other secondary causes of AAV can be challenging due to the heterogeneity of clinical and serologic presentations in PRS secondary to LAC-induced AAV and their substantial overlapping features with primary AAV. A detailed history eliciting any cocaine exposure may be the best and sometimes only clue to separating these entities. Recognizing the diagnosis of LAC-induced AAV is difficult but crucial. The prognosis for PRS secondary to LAC-induced AAV may depend on the patient's understanding and willingness to discontinue cocaine use, although data on cocaine reintroduction and recurrence of PRS was beyond the scope of this review. Although PRS secondary to either LAC-induced AAV or primary AAV are treated with similar immunosuppressive regimens, it is necessary to identify LAC-induced AAV in order to appropriately counsel on cocaine cessation to improve the chance of recovery and avoid recurrence. Institutional Review Board Statement: Ethical review and approval were waived for this study due to this study NOT constituting human subjects research per an Augusta University Institutional Review Board determination.